Acute alterations in muscle flap microcirculation during tumor necrosis factor alpha-induced inflammation

In reconstructive microsurgery of free tissue transfer, ischemia-reperfusion (IR) injury is an unavoidable component of the procedure, which can affect free flap survival markedly. A notable amount of evidence implicates neutrophils in IR injury. Transforming necrosis factor alpha (TNF-alpha) is known to have a central role as a mediator of neutrophil activation in IR injury. The effect of inflammatory stimuli, TNF-alpha, on flow hemodynamics and leukocyte-endothelial interactions in the muscle flaps submitted to IR injury was investigated. In group 1, 6 rats were administered 1 ml of vehicle solution. In group II rats (N = 6), 1 ml of recombinant human TNF-alpha (10 ng per milliliter) was injected intra-arterially. After an hour of ischemia, the cremaster muscle flaps were monitored at 1-hour intervals during 6 hours of reperfusion. After clamp removal, the number of rolling, adhering, and transmigrating leukocytes in the TNF-alpha group was increased by 4-fold, 3-fold, and 3.5-fold respectively compared with the control group (p < 0.05). The increase in rolling leukocytes continued for as long as 3 more hours, whereas the number of adhering and transmigrating leukocytes remained high throughout the experiment. A significant increase in the diameters of the third- and fourth-order arterioles in the TNF-a group was accompanied by a decrease in the number of flowing capillaries at all intervals (p < 0.05). The effect of TNF-alpha-induced inflammation on leukocyte activation was found to be maximal during the first 3 hours of reperfusion. The vasodilatory effect of TNF-alpha was observed only on the third- and fourth-order arterioles.     

Dose-dependent response to IFN-gamma in muscle flap microcirculation

In this study, the authors attempted to determine the effects of intraarterial administration of various doses of Interferon-gamma (IFN-gamma) on microcirculation in a rat muscle flap model. In Group 1 (control), 0.6 ml vehicle solution-PBS-BSA, in Group 2 0.6 ml IFN-gamma (25 ng/ml), in Group 3 0.6 ml IFN-gamma (50 g/ml), in Group 4 0.6 ml IFN-gamma (100 g/ml), were injected. The diameter of the cremaster arterioles and venules, red blood cell velocities, the number of rolling leukocytes and lymphocytes, sticking leukocytes and lymphocytes, capillary perfusion, and endothelial edema index were evaluated. Deterioration of flow hemodynamics was confirmed by a significant decrease in flow velocity in the main artery (A1) (47 percent in Group 3 and 65 percent in Group 4). All dosages of IFN-gamma caused a statistically significant decrease in rolling leukocytes, but this effect was more obvious in the 25 ng/ml group. Injury to the vascular endothelium was confirmed by a two-fold increase in transmigrating leukocytes in the 100 ng/ml group. This was accompanied by 60 percent and 75 percent drops in capillary perfusion, and by 12 percent and 24 percent drops in the endothelial edema index in Groups 3 and 4, respectively. The results indicate that direct intraarterial administration of IFN-gamma in doses higher than 25 ng/ml may be toxic to muscle flaps.     

The effect of hematoma in muscle flap microcirculation

Free radicals were first mentioned by Healey about the hematoma under skin flaps, trying to explain the blood itself has different effect beyond its space-occupying effect or infection. The aim of this study was to determine whether hematoma would have any effect on neutrophil behavior in muscle flap microcirculation. Rat cremaster muscle flap model was used for measuring intravital different parameters. We used the rat cremaster muscle flap model for direct observation and measurement of rolling and adhesion.The rats were randomized into 6 groups. Groups I and IV: only the cremaster muscle was turned to the abdominal wall (n = 4 and n = 4). Groups II and V: saline was injected under the muscle flap (n = 3 and n = 4). Group III and VI: blood was injected under the muscle flap (n = 4 and n = 5). Two sets of microcirculatory measurements were taken in each group. For the comparisons of the groups, the measurements that were taken at the second hour and at the sixth hour were standardized according to the baseline values of the same groups in the same postcapillary venules. Comparisons within the groups were made by Student t test. Between the groups, analysis of variance (ANOVA) independent test was used.Within groups, the number of adherent neutrophils in the second and sixth hour in hematoma groups were found significantly increased according to the baseline measurements, and the number of rolling neutrophils in the second hour in hematoma group was found increased according to the baseline measurements. Between groups, the number of rolling neutrophils in the second hour was found significantly increased between hematoma and sham/hematoma and control groups and the number of adherent neutrophils in the second and sixth hour was found significantly increased between hematoma and sham/hematoma and control groups.     

Histopathology of muscle flap microcirculation following prolonged ischemia

The purpose of this study was to evaluate histological changes occurring in the microcirculation of a muscle flap following prolonged ischemia and to correlate them with the flow hemodynamics. The cremaster muscle flap model was used for direct in vivo studies of the microcirculation. In 45 rats, vascular clamps were applied to the iliac and femoral vessels following flap isolation. Flaps were subjected to various periods of ischemia, ranging from 4 to 6 hours, and 2 hr of reperfusion. In vivo observations of the microcirculation and vessel diameter measurements were taken at 1 hour intervals for an 8 hr period. With prolonged ischemia, return of circulation to the flap was delayed and no flow was observed following 6 hr of ischemia. Morphologic changes at 6 hr revealed red cell and platelet thrombi formation within the capillaries, marked dilatation of postcapillary venules, endothelial swelling in the capillaries, and microhemorrhage formation around the venules. © 1995 Wiley-Liss, Inc.     

Dose response of enoxaparin at the cremaster muscle flap microcirculation

The effects of different dosages of enoxaparin (Lovenox), a low molecular-weight heparin, on microcirculation were investigated. The cremaster muscle model for intravital microscopy was used. Four groups were studied: in group I (n = 6), the controls no agent was given; in group II (n = 6), enoxaparin (2 mg/kg s.c.), in group III, (n = 6), enoxaparin (4 mg/kg s.c.); and in group IV, (n = 6), exoxaparin (8 mg/kg s.c.). These agents were injected before muscle dissection. All animals were observed under intravital microscopy, and measurements of capillary density and red blood cell velocity were taken at 2, 3, 5, and 7 h following subcutaneous enoxaparin injection. Statistical analysis revealed that the capillary density significantly increased in group II and group III, respectively, (by 33% (P < 0.0001) and 25% (P < 0.01) when compared to group I at the fifth hour. Group IV was not significantly different from group I in capillary density. There was no significant difference in red blood cell velocity in any of the groups. Propensity for bleeding was not observed in any of the groups during the dissections and observation periods except in group IV. In conclusion subcutaneous administration of 2 mg/kg enoxaparin improves (by 33%) capillary density without any bleeding complications at the cremaster muscle flap microcirculation at the fifth hour following injection (P < 0.0001). (c) 2005 Wiley-Liss, Inc. Microsurgery 25:147-151, 2005.     

静脉淤血皮瓣的微循环研究

目的 探讨静脉淤血皮瓣微循环的变化规律.方法 选择兔耳建立静脉淤血的皮瓣模型,采用活体微循环动态观察的方法,观察观察静脉淤血组织内部微循环的变化.结果 皮瓣静脉淤血后,其原有微血管的数目逐渐减少,微血流的速度逐渐减慢,红细胞聚集和白色微血栓形成的情况逐渐加重,72 h皮瓣原有的微循环系统基本衰竭.静脉淤血后36 h,可以观察到有新生微血管的出现;48 h,可以观察到较清晰的新生微血管.观察到新生微血管的淤血组织最终成活,而未观察到新生微血管的淤血组织最终坏死.结论 对于发生静脉淤血的皮瓣,如果不及时解除淤血的原因,其原有的微循环系统呈不可逆的进行性恶化,而微血管的新生对淤血组织的成活起了重要作用.     

Quadriceps femoris muscle flap: largest muscle flap model in the rat.

The purpose of this paper is to present a new muscle flap in the rat: the quadriceps femoris muscle flap based on a pedicle consisting of the femoral vessels. In order to establish the anatomic details of this model, seven rats were explored bilaterally, and the regional anatomy of the thigh was examined. The technical aspect of the model was established by the unilateral harvesting of 18 quadriceps femoris muscles. Findings were that this muscle is primarily supplied by a muscular branch originating at the superficial circumflex iliac artery. The average muscle weight was 6 g and the average pedicle length with femoral vessels was 6 mm. Eight of the harvested flaps were transplanted to the contralateral thigh, and the pedicle was anastomosed to the femoral vessels. The other ten flaps were resutured back to their beds. At 72 hr postoperatively, all flaps were viable with the exception of one of the transplanted flaps which was found to be necrotic. The quadriceps femoris muscle flap is technically both a reliable and simple model. With an average weight of 6 g, this flap is by far the largest described in the rat, and offers a convenient model for testing flap-related techniques and outcomes.     

静脉瘀血皮瓣的微血管研究进展

静脉瘀血是各种皮瓣移植术后常见的并发症之一,如果静脉瘀血未被及时纠正,就会危及皮瓣的成活,称为皮瓣的静脉危象。有研究显示,静脉危象是导致皮瓣坏死的主要原因,术后早期监测微循环是预防和处理皮瓣血管危象重要的措施。本文就静脉瘀血皮瓣的微血管研究进展综述如下。     

Late effects of TNF-alpha-induced inflammation on the microcirculation of cremaster muscle flaps under intravital microscopy

In order to understand the microcirculatory changes and regulatory mechanisms governing passage of neutrophils from the vascular bed to the interstitial tissue during ischemia/reperfusion (I/R) injury, a key component of this injury, tumor necrosis factor-alpha (TNF-alpha)-induced inflammation was analyzed. Twenty-four Sprague-Dawley rats were divided into four groups, containing six animals in each. The effect of TNF-alpha-induced inflammation was studied at two different time points, early sequential and late. In the early-effect Groups 1 and 2, animals were given TNF-alpha and vehicle, respectively. Microcirculatory changes were recorded for 6 hr continuously. In the late-effect Groups 3 and 4, following TNF-alpha injection and vehicle, microcirculatory changes were measured 16 hr later. In the early-effect groups, the number of rolling and adhering leukocytes was increased immediately following TNF-alpha injection and remained elevated for the first 3 hr (p<0.05). The number of transmigrated leukocytes remained significantly increased throughout the first 6 hr (p<0.05) and returned to normal at 16 hr. In delayed-effect groups, a second peak in the number of rolling leukocytes was noted at 16 hr (p<0.05). The numbers of rolling and adhering lymphocytes, although remained at the baseline for the first 6 hr, was increased 2- and 1.5-fold at 16 hr, respectively (p<0.05). The number of perfused capillaries gradually decreased over time in the TNF-alpha-induced inflammation groups. A vasodilatory response was noted at the third and fourth order arterioles within the first 3 hr of measurement (p<0.05), but returned to normal afterward. The detrimental effects of TNF-alpha-induced inflammation during I/R injury could be prolonged up to 16 hr at the microcirculatory level of the muscle flaps.     

Arterial crush injury causes decrease in tissue perfusion at the level of the microcirculation in skeletal muscle flap

This study was designed to evaluate the effects of crush injuries to the feeding arteries of a muscle flap on microcirculatory haemodynamics. Eighteen male Sprague-Dawley rats were divided into three experimental groups for intravital microscopy of the cremaster muscle flap. Group 1 served as control. In group 2 the common iliac artery and in group 3 additionally the lower abdominal aorta was crushed with a Kocher clamp (17.4 N) over 5 min. Microcirculatory parameters (red blood cell velocity, vessel diameter, and capillary perfusion) were monitored before and 2 h after crush. In the one-level crush group, red blood cell velocities significantly decreased by 39.17% (P=0.046) in first order arterioles and by 32. 91% (P=0.0106) in second order arterioles. In capillary perfusion, a drop of 48.02% (P=0.0039) was noted. In the two-level crush group, red blood cell velocities significantly dropped over 32.06% (P=0. 0250) in first order arterioles, 35.91% (P=0.0065) in second order arterioles, and 45.69% (P=0.0782) in first order venules. Capillary perfusion was reduced by 20.16% (P=0.374). Arterial crush injuries as possible thrombogenic insults may result in a significant decrease in skeletal muscle perfusion although the blood supply through the crushed supplying vessel is maintained.     

Rectus femoris muscle flap donor-site morbidity.

Donor-site morbidity in four patients after reconstruction with free neurovascular rectus femoris muscle was examined through a series of strength tests in which the leg with rectus femoris muscle harvested was compared with the contralateral leg with an intact rectus femoris muscle. The tests were conducted with three testing devices: (1) the 'Con-Trex Leg-press' in which the force and power of right and left leg extensions at 0.2 and 0.4 m/s in a knee angle from 50 to 90 degrees were tested separately; (2) the isometric power tester, which enabled the unilateral evaluation of the isometric leg extension at three knee angles: 50, 70 and 90 degrees ; and (3) at the 'SP-Force Platforms' in which the patients performed a counter-movement jump where the amplitude of the ground reaction force, the parameters maximum force, and the jump height were calculated in order to compare the right and left leg during a single dynamic movement. Our results showed that the patients (with one exception) demonstrated a balanced relationship between the donor leg and the intact contralateral leg. The patient that primarily demonstrated a large strength deficit was retested 3 months later and showed, after an extensive rehabilitation and training program, an impressive increase in strength. The authors concluded that there is no significant limitation in the strength of the donor leg after removal of the rectus femoris muscle and consequently no significant functional donor-site morbidity. We believe that for the realisation of such results that the intraoperative linking of the vastus lateralis muscle with the vastus medialis muscle, especially in their lower third, and an extensive postoperative rehabilitation and training program are essential.     

Local muscle flap in a venous hypertensive environment.

Local rotation muscle flaps are useful for soft tissue reconstruction in open grade III fractures of the lower extremity. Gastrocnemius or soleus muscle flaps provide early bone coverage and avoid a more demanding free tissue transfer. Deep vein thrombosis is common in injured limbs and venous hypertension may result in acute muscle flap necrosis. Loss of bone coverage and an increased risk of osteomyelitis follow. We present a successful soleus rotation flap for tibial coverage in a lower extremity with a documented major venous thrombosis.     

Late-preconditioning protection is evident in the microcirculation of denervated skeletal muscle.

We investigated whether ischemic preconditioning induces microvascular protection in skeletal muscle at the late phase (after 24 hours) when the same muscles are subjected to prolonged warm global ischemia. The cremaster muscle of the male Sprague-Dawley rat underwent vascular isolation and was subjected to 4 hours of ischemia and 60 minutes of reperfusion. Early preconditioning consisted of 45 minutes of ischemia followed by 15 minutes of reperfusion before prolonged ischemia/reperfusion; late preconditioning also consisted of 45 minutes of ischemia but was done 24 hours (24-hour period of reperfusion) before the prolonged ischemia/reperfusion. Arteriole diameters and capillary perfusion were measured with use of intravital microscopy. Four groups were compared: rats that underwent early preconditioning, their controls, rats that underwent late preconditioning, and their controls. Early and late preconditioning significantly attenuated vasospasm and capillary no-reflow compared with the controls for each. Average arteriole diameter was significantly larger in the rats that underwent late preconditioning than in any other rats; it was also significantly larger in the controls for late preconditioning than in those for early preconditioning. We introduce a model of the rat cremaster muscle that has been isolated from its vascular supply as a useful preparation to study the effects of late preconditioning on microcirculation in skeletal muscle. Late preconditioning provided better microvascular protection than did early preconditioning. The mechanism for this preconditioning protection is being investigated because it should provide a means for therapeutic intervention.     

Intermittent pneumatic compression of legs increases microcirculation in distant skeletal muscle.

Intermittent pneumatic compression has been established as a method of clinically preventing deep vein thrombosis, but the mechanism has not been documented. This study observed the effects of intermittent pneumatic compression of legs on the microcirculation of distant skeletal muscle. The cremaster muscles of 80 male rats were exposed, a specially designed intermittent pneumatic-compression device was applied to both legs for 60 minutes, and the microcirculation of the muscles was assessed by measurement of the vessel diameter in three categories (10-20, 21-40, and 41-70 microm) for 120 minutes. The results showed significant vasodilation in arterial and venous vessels during the application of intermittent pneumatic compression, which disappeared after termination of the compression. The vasodilation reached a maximum 30 minutes after initiation of the compression and could be completely blocked by an inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine (10 micromol/min). A 120-minute infusion of NG-monomethyl-L-arginine, beginning coincident with 60 minutes of intermittent pneumatic compression, resulted in a significant decrease in arterial diameter that remained at almost the same level after termination of the compression. The magnitude of the decrease in diameter in the group treated with intermittent pneumatic compression and NG-monomethyl-L-arginine was comparable with that in the group treated with NG-monomethyl-L-arginine alone. The results imply that the production of nitric oxide is involved in the positive influence of intermittent pneumatic compression on circulation. It is postulated that the rapid increase in venous velocity induced by intermittent pneumatic compression produces strong shear stress on the vascular endothelium, which stimulates an increased release of nitric oxide and thereby causes systemic vasodilation.     

Intravascular thrombosis in skeletal muscle microcirculation after ischemia

This study was undertaken to elucidate the role of thrombosis in blood vessels less than 0.1 mm in diameter in skeletal muscle after an ischemic episode. Capillaries were examined after normothermic ischemia using intravital video microscopy of the cremaster muscle of an anesthetized rat. Histologic sections of the cremaster muscle and silicone rubber intravascular casts were also analyzed. Our model demonstrates that, after 3 hours of warm ischemia, up to two-thirds of capillaries are clotted, as is most of the venous system. Our findings also indicate that capillaries may reopen during 30 minutes of reperfusion. These findings suggest that ischemia may cause thrombosis in the microvasculature of skeletal muscle but the thrombosis appears to be partially reversible.     

Endothelin receptor blockade improves oxygenation in contralateral TRAM flap tissue in pigs.

Partial skin and fat necrosis is the most common complication occurring in TRAM flaps. It is related to disturbances of the microcirculation and oxygenation in the contralateral part of the flap. It may be hypothesised that the development of necrosis is promoted by the vasoconstrictor endothelin, the production of which is enhanced in ischaemic flap tissues. The purpose of this study was to evaluate the effect of tezosentan, a new endothelin receptor blocker, on microcirculation and oxygenation in experimental TRAM flaps. The administration of tezosentan began preoperatively (3 mg/kg body weight) and then continued at a rate of 1.5 mg/kg/h. A TRAM flap with a skin island measuring 16 x 8 cm was raised in the middle of the epigastrium in minipigs. The flap was pedicled on the right superior epigastric vessels. Microcirculatory blood flow was measured with laser Doppler flowmetry and tissue oxygen tension was measured with a Clark-type microprobe. Dominant subcutaneous veins were cannulated in both the ipsilateral and the contralateral parts of the flap. Subdermal tissue oxygen tension in the contralateral part of the flap was significantly reduced 4h after surgery to 5 mmHg (ca. 48 mmHg in normal tissue) in the control group, but to only 12 mmHg in the group that had been administered tezosentan (P< 0.05). Furthermore, tezosentan significantly attenuated venous hypertension (14 mmHg versus 24 mmHg), as well as lactate (4.0 mmol/l versus 5.6 mmol/l) and haemoglobin (10.2 g/dl versus 11.4 g/dl) concentrations in the venous effluent of the contralateral part of the flap, although microcirculatory blood flow remained virtually unchanged. Our findings suggest that tezosentan improves oxygenation and metabolism in the jeopardised contralateral flap tissue, probably as a result of a decrease in venous vascular resistance and fluid extravasation.